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Supplemental Material
Supplemental Methods
Quantification of Germ Cell Number
L4 stage worms were incubated at 25˚C for 24 hours and then washed from plates in M9
buffer. Pelleted worms were subsequently fixed in methanol for at least 5 minutes at 20˚C. Fixed worms were washed once with phosphate-buffered saline containing 0.1%
v/v Tween-20 (PBST), stained with 1 μg/mL DAPI (4',6-diamidino-2-phenylindole) in
PBST for 30 minutes in the dark, cleared of excess DAPI by one wash in PBST, and then
mounted directly onto glass slides. Pachytene nuclei were identified in whole worms by
their distinctive chromosomal morphology using standard epifluorescence filters.
Scanning images throughout the entire thickness of the germline were recorded with a
Hamamatsu CCD camera and total pachytene nuclei in one gonad arm of each worm
were quantified using Openlab software (PerkinElmer).
Quantification of Apoptotic Cell Engulfment in pdk-1(mg142)
L4 stage worms were irradiated with 60 Gy and then incubated for 24 hours at 25˚C. At
this point, wild-type and pdk-1(mg142) worms were mounted abreast on a single slide
and serial images were taken through the entire thickness of the gonad using recorded set
points in the Openlab software. Images were acquired every 15 minutes for 2.5 hours.
The latency of an apoptotic cell began the moment that changes in refractivity made it
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distinguishable from neighbours and ended upon disappearance of the nucleus. Ten
apoptotic cells of each genotype were examined to obtain the average latency in a given
strain.
Germline Immunostaining
Staining was carried out using the methods described in Materials and Methods. To
detect CED-4 or CED-9, germlines were stained with 1:300 dilution of rabbit α-CED-4
(9104) [1] or 1:100 dilution of rabbit -CED-9 (a gift from Dr. B. Conradt), and a 1:200
dilution of mouse Nuclear Pore Complex Proteins mAb414 (Abcam) or 1:150
mitochondrial ATPase ATP5a (Abcam) antibodies overnight in a humid chamber with
parafilm covering the germlines. SIR-2.1 (a gift from Dr. A. Gartner) was detected using
the same method, except rabbit α-SIR-2.1 was diluted 1:250 in PBST [1].
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Supplemental Tables
Supplemental Table 1. daf-2 and pdk-1 do not regulate germ cell proliferation
Genotype
Number of Pachytene Nuclei ± SD N
wild-type
334 ± 49
10
daf-2(e1370)
268 ± 37
10
pdk-1(sa680)
260 ± 67
10
pdk-1(mg142)
335 ± 111
10
daf-16(mgDf47); daf-2(e1370)
315 ± 55
10
Young adult worms (24 hours post-L4 stage at 25ºC) were methanol-fixed and stained
with DAPI to identify pachytene nuclei. For each animal observed, only one gonad arm
was scored. SD = standard deviation.
Supplemental Table 2. Loss of pdk-1 cannot rescue the sterility of ced-9(0) worms
Brood Size (N=3) % Eggs Hatching % Reaching L4/Adult
Genotype
Wild-type
272.8
99.82
100
†
pdk-1(sa709)
107
100
100
ced-9(n2812)
7.7
0
0
ced-9(n2812);
0
NA
NA
pdk-1(sa709)
†
pdk-1(sa709) displays a serotonin-insensitive, partially penetrant Egl phenotype that
results in the death of adults from internal hatching of progeny before the entire brood is
laid.
NA = Not applicable
Worms were transferred to freshly seeded plates every 12 hours beginning at the L4
stage. Eggs laid were quantified immediately after transfer and the percentage of these
that hatched was determined 24 hours later. The number of F1 progeny reaching
L4/Adulthood was counted when synchronously laid wild-type progeny reached this
point.
Supplemental Table 3. pdk-1 does not regulate the clearance of apoptotic cells
Genotype
Wild-type
pdk-1(mg142)
Average Corpse Latency ± SD (min)
48.0 ± 24.3
49.1 ± 31.5
N
10
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L4-stage worms were treated with 60 Gy of ionizing radiation and then incubated at 25ºC
for 24 hours. Persistence of apoptotic cells (corpses) was monitored by sequential
microscopy, using the onset of refractivity as the initiating event and the disappearance of
the cell as the closing.
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Supplemental Figure Legends
Supplemental Figure 1. daf-2 and pdk-1 do not affect physiological germ cell
apoptosis.
L4 stage worms were incubated for 24 hours at 20°C and germ cell apoptosis in the
absence of irradiation was quantified. Error bars as in Figure 1.
Supplemental Figure 2. akt-1(ok525) is a protein-null allele.
Young adult worms were lysed and soluble AKT-1 was immunoprecipitated. After
separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were
blotted to polyvinylidene fluoride (PVDF) and probed with a 1:1 mixture of AKT-1 128
and 527 antisera.
Supplemental Figure 3. Endogenous CED-9 protein is not altered in the germlines of
irradiated daf-2 mutants. Germlines were isolated from young adult stage worms 24
hours after exposure to 120 Gy IR and stained with antibodies to CED-9 (a gift from
Barbara Conradt) and mitochondrial ATPase (ATP5A, Abcam), and nuclei were stained
with DAPI, as described in Materials and Methods. Stained germlines were mounted on
slides and visualized by epifluorescence microscopy. daf-2(lf) = daf-2(e1370), ced-9(0);
ced-3(0) = ced-9(n2812); ced-3(n717). Bar = 35 μm.
Supplemental Figure 4. Neither IR nor pdk-1 status affect CED-4 localization in the
germline.
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A) CED-4 localizes at the nuclear periphery (arrows) and this does not change with IR or
in pdk-1 mutants. Bright puncta in pdk-1(gf) mutant germ cells at 120 Gy were not
reproducible in replicate experiments. CED-4 localization is not altered in actively dying
cells (triangle), as determined by condensed chromatin in pdk-1(gf) mutants. In the
terminal phases of cell death, CED-4 expression is lost in concert with the nuclear
envelope (chevrons). Worms were irradiated at the L4 stage and germlines were
extruded after 24 hours at 25ºC. Dissected germlines were fixed and stained with
antibodies against CED-4 [1] and the nuclear pore complex, as described in Materials and
Methods, followed by DNA staining with DAPI. Stained germlines were mounted on
slides and visualized by epifluorescence microscopy. pdk-1(0) = pdk-1(sa680), pdk-1(gf)
= pdk-1(mg142), ced-4(0) = ced-4(n2273) – a null allele [2]. Bar = 10 μm.
B) Nuclear PDK-1 intensity in Figure S2A was quantified using ImageJ, normalized
against nuclear pore complex intensity in the same section and the normalized PDK-1
nuclear intensity was subsequently expressed relative to wild-type unirradiated. Error
bars represent the standard deviation from at least ten nuclei.
C) SIR-2.1 localizes to the nucleus in most live cells (left panels; arrows). A small subset
of live cells lack nuclear SIR-2.1 (left panels; arrowheads). These cells are not apoptotic
and the proportion of SIR-2.1-negative live cells did not differ between genotypes. SIR2.1 did not change localization following IR except in apoptotic cells where it was lost
from the nucleus in all strains examined (right panels; chevrons). Worms were irradiated
as young adults and germlines were extruded after 24 hours at 20ºC. Dissected germlines
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were fixed and stained with antibodies as described in [1]. Stained germlines were
mounted on slides and visualized by epifluorescence microscopy. pdk-1(lf) = pdk1(sa709), pdk-1(gf) = pdk-1(mg142), daf-2(lf) = daf-2(e1370), sir-2.1(lf) = sir2.1(ok434). Bar = 10 μm.
Supplemental References
1. Greiss S, Hall J, Ahmed S, Gartner A (2008) C. elegans SIR-2.1 translocation is
linked to a proapoptotic pathway parallel to cep-1/p53 during DNA damage-induced
apoptosis. Genes Dev 22: 2831-2842.
2. Yuan J, Horvitz HR (1992) The Caenorhabditis elegans cell death gene ced-4
encodes a novel protein and is expressed during the period of extensive programmed
cell death. Development 116:309-320.
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